Methods for producing chiral chromones, chromanes, amino substituted chromanes and intermediates therefor

ABSTRACT

Disclosed are process steps and novel processes for producing chromane compositions enriched in at least one (2R or 2S) enantiomer, preferably chroman-2-yl carboxylic acid compounds and chroman-2-yl carboxylic acid esters which are intermediates for producing platelet aggregation inhibitors and/or are themselves potent platelet aggregation inhibitors. Further disclosed are enzymatic processes for resolving chiral intermediates or final products to provide desired enantiomers.

This is the U.S. national phase under 35 U.S.C. §371 of Internationalapplication PCT/US01/17980, published in English, filed Jun. 1, 2001,which claims priority to U.S. Provisional Application No. 60/208,827,filed Jun. 2, 2000.

FIELD OF THE INVENTION

This invention relates to novel processes for producing chromanecompounds, preferably chroman-2-yl acetic acid compounds and aminosubstituted chroman-2-yl acetic acid esters which are intermediates forproducing platelet aggregation inhibitors and/or are themselves potentplatelet aggregation inhibitors. It further relates to processes forresolving chiral intermediates or final products to provide desiredenantiomers.

BACKGROUND OF THE INVENTION

One process for making chromanes from coumarin derivatives is describedin U.S. Pat. No. 5,731,324 at pages 101-103. The unprotected aminoderivative bicyclic compound is shown on page 147. However, that processinvolves chromatography as a purification step, which does not scalewell commercially.

SUMMARY OF THE INVENTION

In accordance with one preferred embodiment, there is provided a processfor making a compound, or a salt thereof, having a general formula:

wherein R is C₁-C₈ alkyl and n=0 to about 3. The method comprises (a)through (f) below:

(a) reacting phenol and beta-keto glutaric add in H₂SO₄/Ethanol withheat, followed by pouring the reaction mixture onto ice water,extracting into organic solvent and evaporating as follows:

(b) hydrogenating the chromenone product from (a) above to produce thecorresponding chromanone:

(c) nitrating the chromanone from (b) as follows:

(d) resolving the racemic mixture using a lipase enzyme, as follows:

(e) hydrogenating the 4-carbon to remove the oxo group and convert thenitro group to an acetamido group as follows:

(f) acidifying the product from (e) above to recover the amine followedby addition of concentrated HCl to produce the HCl salt as follows:

In accordance with one preferred embodiment, there is provided a processfor making a compound, or a salt thereof, having a general formula:

wherein R is C₁-C₈ alkyl and n=0 to about 3. The method comprises (a)through (g) below:

(a) reacting 2-hydroxyacetophenone and diethyloxalate in the presence ofsodium ethoxide followed by addition of concentrated sulfuric acid tomake the bicyclic ring system as follows:

(b) hydrogenating the chromen-4-one to form the chromen-4-one asfollows:

(c) performing a chain extension by first making the free acid, followedby reacting with borane-methyl sulfide complex to form the2-hydroxymethyl derivative, followed by replacing the hydroxy group witha tosyl group and reacting the tosyl derivative with a cyanide salt toform a 2-cyano derivative, followed by acidifying the cyano derivativein concentrated acid and esterifying the 2-acid group as follows:

(d) nitrating the product from (c) above to form the 6-nitro group asfollows:

(e) resolving the racemic mixture using a lipase enzyme, as follows:

(f) hydrogenating the 4-carbon to remove the oxo group and convert thenitro group to an acetamido group as follows:

(g) acidifying the product from (f) above to recover the amine followedby addition of concentrated HCl to produce the HCl salt as follows:

In accordance with one preferred embodiment, there is provided a processfor making a compound, or a salt thereof, having a general formula:

wherein R is C₁-C₈ alkyl and n=0 to about 3. The method comprises (a)through (e) below:

(a) reacting nitrophenol and diethyl ester of maleic add with methanesulfonic acid under heating as follows:

(b) performing a chain extension by first making the free aid, followedby reacting with borane-methyl sulfide complex to from the2-hydroxymethyl derivative, followed by replacing the hydroxy group witha tosyl group and reacting the tosyl derivative with a cyanide salt toform a 2-cyano derivative, followed by acidifying the cyano derivativein concentrated acid and esterifying the 2-acid group as follows:

(c) resolving the racemic mixture using a lipase enzyme, as follows:

(d) hydrogenating the 4-carbon to remove the oxo group and convert thenitro group to an acetamido group as follows:

(e) acidifying the product from (d) above to recover the amine followedby addition of concentrated HCl to produce the HCl salt as follows:

In accordance with one preferred embodiment, there is provided a processfor making a compound, or a salt thereof, having a general formula:

wherein R is C₁-C₈ alkyl and n=0 to about 3. The process comprises (a)through (g) below:

(a) nitrating the chromen-4-one at the 6-position as follows:

(b) reacting the product from (a) above with TBSOTf to form abenzopyrillium salt as follows:

(c) adding the ketene enol to the benzopyrillium salt from (b) above asfollows:

(d) acidifying the product from (c) above to complete the addition ofthe substituent at the 2-position on the 6-nitro-4-oxochromane ring asfollows:

(e) resolving the racemic mixture using a lipase enzyme, as follows:

(f) hydrogenating the 4-carbon to remove the oxo group and convert thenitro group to an acetamido group as follows:

(g) acidifying the product from (f) above to recover the amine followedby addition of concentrated HCl to produce the HCl salt as follows:

The compositions formed according to the above methods preferablycomprise about 75% to about 100% of a single (2R) or (2S) enantiomer of6-aminochroman-2-yl acetic acid or an ester thereof.

In preferred embodiments, the lipase is from Pseudomonas cepacia, is thePS 30 lipase, is stabilized by cross-linking with alpha keto glutarateand the like, or is the stabilized PS 30 enzyme ChiroCLEC-PC.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In view of the shortcomings of the known process mentioned above, thereis a need for improved processes for producing compounds that are usefulas intermediates in processes for producing platelet aggregationinhibitors. There is a particular need for Improved processes for makingcompounds having the phenyl ring of the benzopyrans substituted by anamino group or a protected amino group. Such intermediates are usefulfor coupling with a carbonyl group to produce a carboxamide link andresult in compounds that are useful platelet aggregation inhibitors orintermediates for forming platelet aggregation inhibitors. Also neededis a process to produce relatively inexpensively large quantities ofchromone intermediates that are useful for being resolved byconventional processes to produce benzopyran or chromane derivativeswherein the chiral center at the two position of the saturated pyranring portion of the bicyclic ring structure can be resolved into racemicmixtures (R/S) that are enriched with one of the R or S enantiomers orto produce substantially pure compositions of a single enantiomer (R orS enantiomer). Due to inherent losses of up to 50% or more of thestarting materials (assuming a 50/50 R/S racemate) during enantiomericresolution, there is a need for a process which is efficient enough tobe scaled to an industrial level for inexpensively producing largequantities of a desired intermediate compound or large quantities offinal chroman-2-yl acetic acid ester compounds that are useful in theanticoagulant field.

Accordingly, there continues to be a need for a process that isadaptable to commercially scaleable production of such chromanes. One ormore of the foregoing needs may be met using the processes describedherein and the compounds and intermediates made thereby.

The disclosure herein presents novel processes for producing chromanecompounds, preferably chroman-2-yl acetic acid compounds and aminosubstituted chroman-2-yl acetic acid esters which are intermediates forproducing therapeutic agents, or are themselves therapeutic agents, fordisease states in mammals that have disorders caused by or Impacted byplatelet dependent narrowing of the blood supply.

In particular, disclosed are processes that utilizes a Simonis Chromonecyclizing step with a phenol starting material and glutarate ketoneunder acidic condition (P₂O₅, phosphorous oxychloride or H₂SO₄).Preferably, sulfuric acid and absolute ethanol are utilized to producethe acetic add ethyl ester at the 2-position as follows:

wherein R is a substituent on the phenyl or benzene ring and R is anitro group or an amino group (or a protected amino group such as abenzamido or acetamido group) or a group that can be converted to anitro or amino group (e.g., hydrogen or halogen). Preferably, the2-carboxylic acid group is esterified with an ethyl group and the Rgroup on the benzene or phenyl portion is hydrogen, halogen or a NO₂group Most preferably, the R group is a hydrogen atom.

The double bond of the oxo-pyran portion of the bicyclic ring ispreferably reduced by hydrogenation processes such as hydrogen and Pd/C,or the like as follows:

to produce the chromone product from the chromenone compound. Thepresence of ethyl acetate, ethanol and the like as a solvent canminimize the formation of a hydroxyl group from the carbonyl group inthe ring. In any event, a standard reaction to covert a hydroxyl groupto a ketone (carbonyl) may be alternatively utilized if a hydroxyl groupis desired. The resulting compound can be substituted with a desired Rsubstituent in the phenyl ring, preferably with a 6-position NO₂ group,by reacting the chromone compound with a nitrating agent such as ametallic nitrate in a mineral acid. Preferably, potassium nitrate isutilized with sulfuric acid at a temperature of from 0° C. to roomtemperature, but any. nitration procedure may be used. Due to steric andelectronic directing, a very high amount of the 6-position nitrocompound is obtained and the reaction may be monitored with HPLC, forexample, to determine completion. Work-up is done by crystallization,e.g., ethyl acetate or toluene, to favor a particular position isomer.

The nitrating reaction may be illustrated as follows:

to provide a racemic ethyl (6-nitro-4-oxochroman-2-yl)acetate as shownabove. Also, as is clear from the above discussion, If R is a nitrogroup prior to the hydrogenation step, an amino group on the phenyl ringresults from the hydrogenation with the Pd/C, or the like. Since certainlipases may favor the hydrolysis of the acetic acid ester when the nitrogroup is present on the phenyl ring, it is preferred that the nitrogroup be added to the phenyl ring after the hydrogenation step iscompleted. The 6-position racemates, including the preferred 6-nitrocompound, may be generically illustrated as follows:

wherein R is a nitro group, an amino group or a protected amino group,such as an acetamido or benzamido group. The individual enantiomers ofthe racemate can be resolved as set forth below.

Alternatively, to avoid a hydrogenation step prior to nitration, theracemate set forth above can be produced via a benzopyrilium salt bytreating a 6-nitro-4-oxo-2-chromene nucleus with TBSOTf, and subjectingthe resulting intermediate to a silyl enol ether hydrolysis. The6-nitro-chromone nucleus is available commercially or can be produced,for example, by nitrating the chromone (4-oxo-2-chromene) at roomtemperature (Aldrich Catalog Number 19922-2). The nitration step may beillustrated as follows:

to selectively produce the 6-nitro-chromone nucleus in a high yield,since the electronic nature of chromone nucleus favors the placement ofthe nitro group in the 6 position on the ring. A benzopyrilium salt isthen produced from this chromone nucleus as follows:

Further, the silyl ketene acetal reactant can be produced from theacetic acid ethyl ester and TBSOTf by using standard methods in thesilyl ketene acetal art and reacted with the benzopyrilium salt that isset forth above, which was obtained from the 6-nitrochromoneintermediate. Reaction of the salt and the silyl ketene acetal areillustrated as follows:

Acidifying the above compound with aqueous HCl, or the like results inthe desired racemate, 6-nitro-4-oxochroman-2-yl-acetic acid (ethylester), which can be resolved in the same manner as the racemateproduced by the Simonis Chromone cyclization procedure, see below.

The overall yield from the benzopyrillium salt to the racemate as setforth in the above alternative to the Simonis Chromone cyclizationprocedure is quite good, and is usually from about 88-95% yield. Oneembodiment, which summarizes the above reaction steps as well asenzymatic resolution of the racemate, is set forth below in Scheme IV.One might note that while the chromone and 6-nitro-chromone compoundsdescribed above are standard Items of commerce, routine methods exist inthe art for producing such starting materials. Other obvious variationsand permutations of the above benzopyrilium salt procedure will bereadily apparent to one of ordinary skill in the art in view of thediscussion herein and are considered to be within the scope of thedisclosure.

A chirally selective lipase such as the Altus, Inc. ChiroCLEC-PC lipase,or the like, may be utilized to resolve the ethyl2-(6-nitro-4-chromanone)acetate racemate, regardless of whether it isproduced by the Simonis Chromone cyclization or via the abovebenzopyrilium salt process. In the case where the nitro compound isutilized 98.5 percent of the acid formed corresponds to one enantiomer.Crystallization, or other standard separation procedures, may beutilized to separate the acid from the ester and result in asubstantially pure or enriched composition of a single enantiomer.Depending upon the desired enantiomer, the crystals or the supernatantmay be chosen to be used for further processing.

The undesired isomer may be recycled by using a racemization stepfollowed by re-exposure of the resulting racemate to the lipase. Theformation of a racemate from a single enantiomer is accomplished byexposing the enantiomer to a basic alcoholic solution such as a sodiumor potassium ethanolate solution. Other procedures which open the ringat the ring oxygen of the chromone and then reclose it may also beutilized to produce a racemate from a single enantiomer. By repeatingthe resolution and racemate forming steps, a higher overall yield may beobtained. The racemate forming step may be illustrated as follows:

wherein, as illustrated, a catalytic amount of potassium carbonate orsimilar catalytic base in R¹OH (preferably EtOH) is utilized for 1-3days at room temperature, saponified with aqueous 1N NaOH in R¹OH for 3hours. After acidification with 1N HCl, the racemic acid may becrystallized out, washed, and re-esterified with an acidic ethanolsolution. The resulting ester racemate can then be recycled by exposureto the lipase to obtain a higher yield of the desired single enantiomerwith respect to the initial amount of racemate starting material.

After the resolution of the enantiomers, the 4-oxo group can be removedfrom the (R) or (S) enantiomer chromone compound and the R group can beconverted to an amino group by a simple hydrogenation reaction.Preferably, hydrogen gas and a Pd/C catalyst, or the like, are used toproduce a substantially pure single enantiomer of a (2 R or S)chroman-2-yl)acetic acid ester having the phenyl portion substituted asindicated above,: preferably in the 6-position. For example, glacialacetic acid and 30-60 psi of hydrogen at 40-80° C. in the presence of acatalyst such as palladium on carbon may be utilized in a hydrogenator.The reaction can be monitored with HPLC to determine the completion ofthe hydrogenation. Molecular sieves may optionally be used as well.

Such a hydrogenation reaction is exemplified as follows:

wherein, the amino group can be in the form of a mineral acid salt byaddition of an acidic alcohol solution to the compound. Further, if thehydrogenation reaction results in creation of the free acid, it can beconverted, to the desired ester by treatment with an appropriateester-forming alcohol in sulfuric acid followed by exposure to a mineraladd alcohol solution to provide the mineral acid salt of the aminogroup.

Non-limiting Illustrative Scheme I, set forth below, comprises theprocess steps outlined directly below which may also include furtherinitial starting steps to produce the starting materials which arecommercially available or further processing steps which modify theamino group to comprise a desired functional group, such as groupsdescribed in the anti-coagulation field. Amino coupling reactions arewell-known in the art. Moreover, specific steps that are set forth inthe preferred embodiment reaction scheme below are described in theexamples. The reaction products are isolated and purified byconventional methods, typically by solvent extraction into a compatiblesolvent Preferred solvents are lower alkane ethers and alcohols; ethylether and isopropyl alcohol are preferred for solvent extraction orrecrystallization procedures. Esters of carboxylic add side groups maybe formed that permit selective separation of the R and S enantiomers byuse of hydrolysis with a lipase, solvent extraction orrecrystallization. The products may be further purified by columnchromatography or other appropriate methods.

Alternatively, a corresponding 6-nitro-4-oxo-chroman-2-yl carboxylicacid compound can be produced and esterified by using a5-nitro-acetophenone starting material and the reaction shown in the J.Med. Chem, Vol. 15, No. 8 (1972) or by nitration of the compounddescribed in the referenced article (see Scheme II, below).Additionally, the 6-nitro-4-oxo-chroman-2-yl carboxylic acid ester canbe made by reacting nitrophenol and the diethyl ester of maleic add, forexample (see Scheme III, below).

For example, the carboxylic acid group of the desired enantiomer can beextended to an acetic acid group by reducing the carboxylic acid sidegroup to form a methanol side chain followed by extending its length viaa potassium cyanate reaction and the like. After the side extensionreaction is completed, the racemate of the 6-nitro-4oxo-chroman-2-ylcarboxylic acid ester can be resolved by the above process utilizing theAltus ChiroCLEC-PC enzyme (or any other acceptable lipase). PS 30 andfunctionally similar enzymes can also be utilized, and then extended toan acetic acid ethyl ester side chain after the resolution.

After resolution, the 4-oxo group is removed and the 6-nitro group isreduced to an amino group by a hydrogenation step. For example, ahydrogenator loaded with the compound in ethanol or acetic anhydride inthe presence of 10% palladium on carbon at 40-60° C. and 40-60 psi ofhydrogen can be used for the reduction step, with or without thepresence of molecular sieves. The progress of the reaction can bemonitored via HPLC.

Additional non-limiting schemes are set forth below:

In other embodiments, the order of some of the reactions in the schemesmay be changed, and additional steps of protecting, deprotecting,nitrating, hydrolyzing, esterifying, and the like may be added to theschemes at various points. Such minor alterations are within the scopeof the disclosure herein. Although the esters shown are primarily ethylesters, other esters may be made, either by use of different solventsand/or reagents in the initial formation reactions or bytransesterificaton.

The starting materials used in the disclosed processes are commerciallyavailable from chemical vendors such as Aldrich, Lancaster, TCI, BachemBiosciences, and the like, or may be readily synthesized by knownprocedures including those present in the chemical literature, or may bemade by using procedures such as indicated above.

Reactions are carried out in standard laboratory glassware and reactionvessels under reaction conditions of standard temperature and pressure,except where it is otherwise indicated, or where use of non-STPconditions for a procedure is known in the art. Some procedures,reactions, and/or workups which are well known in the art or which arereadily available in standard reference texts in the art, includingBeilstein and Fieser and Fieser, may not be presented herein owing totheir stature of being within the knowledge of one of ordinary skill.Further, the above procedures of the claimed invention processes may becarried out on a commercial scale by utilizing reactors and standardscale-up equipment available in the art for producing large amounts ofcompounds in the commercial environment. Such equipment and scale-upprocedures are known to the ordinary practitioner in the field ofcommercial chemical production.

During the synthesis of these compounds, amino or acid functional groupsmay be protected by blocking groups to prevent undesired reactions withthe amino group during certain procedures. Procedures for suchprotection and removal of protecting groups are routine and well knownto the ordinary practitioner in this field.

Enantiomeric Resolution and Acid Salt Formation

When a reaction results in the production of racemic chroman-2-ylcarboxylic acids and esters, these racemates are preferably resolved toproduce a mixture enriched in one of the R or S enantiomers orcompletely resolved into a substantially pure composition of one of theenantiomers. Examples of processes for resolving the racemic mixturesare provided herein and/or are known to those skilled in the artAdditionally, processes for the formation of acid addition salts such asthe hydrochloride salt of the 6-position amino acid group on thechromane nucleus are known in the art. Other such salts are alsoenvisioned.

Uses of Compounds

As mentioned above, the compounds produced according to preferredembodiments find utility as intermediates for producing therapeuticagents or as therapeutic agents for disease states in mammals, includingthose which have disorders that are due to platelet dependent narrowingof the blood vessels, such as atherosclerosis and arteriosclerosis,acute myocardial infarction, chronic stable angina, unstable angina,transient ischemic attacks and strokes, peripheral vascular disease,arterial thrombosis, preeclampsia, embolism, restenosis followingangloplasty, carotid endarterectomy, anastomosis of vascular grafts, andetc. These conditions represent a variety of disorders thought to beinitiated by platelet activation on vessel walls.

Platelet adhesion and aggregation is believed to be an important part ofthrombus formation. This activity is mediated by a number of plateletadhesive glycoproteins. The binding sites for fibrinogen, fibronectinand other clotting factors have been located on the platelet membraneglycoprotein complex IIb/IIIa. When a platelet is activated by anagonist such as thrombin, the GPIIb/IIIa binding site becomes availableto fibrinogen, eventually resulting in platelet aggregation and clotformation. Thus, intermediate compounds for producing compounds thateffective in the inhibition of platelet aggregation and reduction of theincidence of clot formation are useful intermediate compounds.

The compounds produced according to preferred embodiments may also beused as intermediates to form compounds that may be administered incombination or concert with other therapeutic or diagnostic agents. Incertain preferred embodiments, the compounds produced by theintermediates according to the present invention may be co-administeredalong with other compounds typically prescribed for these conditionsaccording to generally accepted medical practice such as anticoagulantagents, thrombolytic agents, or other antithrombotics, includingplatelet aggregation inhibitors, issue plasminogen activators,urokinase, prourokinase, streptokinase, heparin, aspirin, or warfarin.The compounds produced from the intermediates may act in a synergisticfashion to prevent reocclusion following a successful thrombolytictherapy and/or reduce the time to reperfusion. Such compounds may alsoallow for reduced doses of the thrombolytic agents to be used andtherefore minimize potential hemorrhagic side-effects. Such compoundscan be utilized in vivo, ordinarily in mammals such as primates, (e.g.humans), sheep, horses, cattle, pigs, dogs, cats, rats and mice, or invitro.

Coupling Reaction of the Hydrochloride Salt Intermediate Compounds

The above compounds produced according to preferred methods may beisolated and further reacted to substitute a desired group for one ormore of the hydrogen atoms on the amino group by a coupling reaction.Particularly preferred is a coupling reaction of the amino group with anacyl halide compound. For example, compounds such as5-amidino-thiophen-2-yl carboxylic acid derivatives (or an acyl halidesuch as the acyl chloride) and 4-amidinobenzoyl chloride may be coupledto ethyl (2S)-(6-amino-chroman-2-yl) acetate (or its hydrochloride salt)to form ethyl (2S)[6-(5-amidino-2-thiophenoyl)amino-chroman-2-yl]acetateand ethyl (2S)-[6-(4-amidinophenyl) carbonylamino]chroman-2-yl} acetate,or other similar compounds or their derivatives which are known plateletaggregation inhibitors. For examples of such platelet aggregationinhibitors, see U.S. Pat. No. 5,731,324. The ring portion of the aboveamidino-aroyl or amidino-heteroaroyl derivatives may be substituted bygroups such as methyl, ethyl, fluoro, iodo, bromo, chloro, methoxy,ethyoxy, and the like which results in compounds that are known plateletaggregation inhibitors. Standard coupling procedures may be utilized,but procedures utilizing reaction mixtures the compounds, in salt form,are suspended in solvents such as acetonitrile, toluene, or the like,are preferred.

The compound formed from the coupling reaction may be used as either thesalt or the free base, and may be readily interconverted between the twoforms by using procedures which include-those known in the art as wellas reacting the compound with one or more molar equivalents of thedesired acid or base in a solvent or solvent mixture in which the saltis insoluble, or in a solvent like water after which the solvent isremoved by evaporation, distillation or freeze drying. Alternatively,the free acid or base form of the product may be passed over an ionexchange resin to form the desired salt, or one salt form of the productmay be converted to another using the same general process. The freebase or salts may be purified by various techniques such asrecrystallization in a lower alkanol such as methanol, ethanol,propanol, isopropanol and the like, for example, or a mixture thereof.In preferred embodiments, the compound is recovered as the hydrochloridesalt and the recrystallization solvent is a 90/10-10/90 mixture ofethanol and isopropanol. Non-toxic and physiologically compatible saltsare preferred, although other types of salts may also be used, such asin the processes of Isolation and purification.

Compositions and Formulations

Diagnostic and therapeutic applications of the compounds formed byprocedures disclosed herein, including the aforementioned couplingreactions, will typically utilize formulations wherein the compound, ora pharmaceutically acceptable salt, solvate, or prodrug, is combinedwith one or more adjuvants, excipients, solvents, or carriers. Theformulations may exist in forms including, but not limited to tablets,capsules or elixirs for oral administration; suppositories; sterilesolutions or suspensions for injectable or parenteral administration; orincorporated into shaped articles. Subjects in need of treatment(typically mammalian) using the compounds of this invention can beadministered dosages that will provide optimal efficacy. The dose andmethod of administration will vary from subject to subject and bedependent upon such factors as the type of mammal being treated, itssex, weight, diet, concurrent medication, overall clinical condition,the particular compounds employed, the specific use for which thesecompounds are employed, and other factors which those skilled in themedical arts will recognize.

Formulations are prepared for storage or administration by mixing thecompound, or a pharmaceutically acceptable salt, solvate or prodrugthereof, having a desired degree of purity with physiologicallyacceptable carriers, excipients, stabilizers etc., and may be providedin sustained release or timed release formulations. Acceptable carriersor diluents for therapeutic use are well known in the pharmaceuticalfield, and are described, for example, in Remington's PharmaceuticalSciences, Mack Publishing Co., (A. R. Gennaro edit. 1985). Suchmaterials are nontoxic to the recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,acetate and other organic acid salts, antioxidants such as ascorbicacid, low molecular weight (less than about ten residues) peptides suchas polyarginine, proteins, such as serum albumin, gelatin, orimmunoglobulins, hydrophilic polymers such as polyvinylpyrrolidinone,amino acids such as glycine, glutamic acid, aspartic acid, or arginine,monosaccharides, disaccharides, and other carbohydrates includingcellulose or its derivatives, glucose, mannose or dextrins, chelatingagents such as EDTA, sugar alcohols such as mannitol or sorbitol,counter ions such as sodium and/or nonionic surfactants such as Tween,Pluronics or polyethyleneglycol.

Dosage formulations to be used for parenteral administration arepreferably sterile. Sterility is readily accomplished by filtrationthrough sterile membranes such as 0.2 micron membranes, or by otherconventional methods known to those skilled in the art. Formulations arepreferably stored in lyophilized form or as an aqueous solution. The pHof such preparations are preferably between 3 and 11, more preferablyfrom 5 to 9 and most preferably from 7 to 8. It will be understood thatuse of certain of the foregoing excipients, carriers, or stabilizerswill result in the formation of cyclic polypeptide salts. While thepreferred route of administration is by injection, other methods ofadministration are also anticipated such as intravenously (bolus and/orinfusion), subcutaneously, intramuscularly, colonically, rectally,nasally or intraperitoneally, employing a variety of dosage forms suchas suppositories, implanted pellets or small cylinders, aerosols, oraldosage formulations and topical formulations such as ointments, dropsand dermal patches. The compounds are desirably incorporated into shapedarticles such as implants which may employ inert materials such asbiodegradable polymers or synthetic silicones, for example, Silastic,silicone rubber or other polymers commercially available.

The compounds may also be administered in the form of liposome deliverysystems, such as small unilamellar vesicles, large unilamellar vesiclesand multilamellar vesicles. Liposomes can be formed from a variety oflipids, such as cholesterol, stearylamine or phosphatidylcholines.

The compounds may also be delivered by the use of antibodies, antibodyfragments, growth factors, hormones, or other targeting moieties, towhich the compound molecules are coupled. The compounds may also becoupled with suitable polymers as targetable drug carriers. Suchpolymers can include polyvinylpyrrolidone, pyran copolymer,polyhydroxy-propyl-methacrylamide-phenol,polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the plateletaggregation inhibitors may be coupled to a class of biodegradablepolymers useful in achieving controlled release of a drug, for examplepolylactic acid, polyglyolic acid, copolymers of polylactic andpolyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydmpyrans, polycyanoacrylates andcross linked or amphipathic block copolymers of hydrogels. Polymers andsemipermeable polymer matrices may be formed into shaped articles, suchas valves, stents, tubing, prostheses and the like.

Therapeutic compound liquid formulations generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by hypodermic injectionneedle.

Therapeutically effective dosages may be determined by either in vitroor in vivo methods. For each particular compound and formulation,individual determinations may be made to determine the optimal dosagerequired. The range of therapeutically effective dosages will naturallybe influenced by the route of administration, the therapeuticobjectives, and the condition of the patient. For injection byhypodermic needle, it may be assumed the dosage is delivered into thebody's fluids. For other routes of administration, the absorptionefficiency must be individually determined for each inhibitor by methodswell known in pharmacology. Accordingly, it may be necessary for thetherapist to titer the dosage and modify the route of administration asrequired to obtain the optimal therapeutic effect. The determination ofeffective dosage levels, that is, the dosage levels necessary to achievethe desired result, will be within the ambit of one skilled in the artTypically, applications of compound are commenced at lower dosagelevels, with dosage levels being increased until the desired effect isachieved.

A typical dosage might range from about 0.001 mg/kg to about 1000 mg/kg,preferably from about 0.01 mg/kg to about 100 mg/kg, and more preferablyfrom about 0.10 mg/kg to about 20 mg/kg. Advantageously, the compoundsor formulations may be administered several times daily, in a once dailydose, or in other dosage regimens.

Typically, about 0.5 to 500 mg of a compound or mixture of compounds, asthe free acid or base form or as a pharmaceutically acceptable salt orprodrug derivative (including esters), is compounded with aphysiologically acceptable vehicle, carrier, excipient, binder,preservative, stabilizer, dye, flavor etc., as called for by acceptedpharmaceutical practice. The amount of active ingredient in thesecompositions is such that a suitable dosage in the range indicated isobtained.

Typical adjuvants which may be incorporated into tablets, capsules andthe like are a binder such as acacia, corn starch or gelatin, andexcipient such as microcrystalline cellulose, a disintegrating agentlike corn starch or alginic add, a lubricant such as magnesium stearate,a sweetening agent such as sucrose or lactose, or a flavoring agent.When a dosage form is a capsule, in addition to the above materials itmay also contain a liquid carrier such as water, saline, a fatty oil.Other materials of various types may be used as coatings or as modifiersof the physical form of the dosage unit Sterile compositions forinjection can be formulated according to conventional pharmaceuticalpractice. For example, dissolution or suspension of the active compoundin a vehicle such as an oil or a synthetic fatty vehicle like ethyloleate, or into a liposome may be desired. Buffers, preservatives,antioxidants and the like can be incorporated according to acceptedpharmaceutical practice.

The compounds and formulations may be used alone or in combination, orin combination with other therapeutic or diagnostic agents. In certainpreferred embodiments, the compounds and/or formulations may becoadministered along with other compounds typically prescribed for theseconditions according to generally accepted medical practice, such asanticoagulant agents, thrombolytic agents, or other antithrombotics,including platelet aggregation inhibitors, tissue plasminogenactivators, urokinase, prourokinase, streptokinase, heparin, aspirin, orwarfarin. The compounds and formulations can be utilized in vivo,ordinarily in mammals such as primates, such as humans, sheep, horses,cattle, pigs, dogs, cats, rats and mice, or in vitro.

The compounds, selected and used as disclosed herein, are believed to beuseful for preventing or treating a condition characterized by undesiredthrombosis, such as (a) the treatment or prevention of anythrombotically mediated acute coronary syndrome including myocardialinfarction, unstable angina, refractory angina, occlusive coronarythrombus occurring post-thrombolytic therapy or post-coronaryangioplasty, (b) the treatment or prevention of any thromboticallymediated cerebrovascular syndrome including embolic stroke, thromboticstroke or transient ischemic attacks, (c) the treatment or prevention ofany thrombotic syndrome occurring in the venous system including deepvenous thrombosis or pulmonary embolus occurring either spontaneously orin the setting of malignancy, surgery or trauma, (d) the treatment orprevention of any coagulopathy including disseminated intravascularcoagulation (including the setting of septic shock or other infection,surgery, pregnancy, trauma or malignancy and whether associated withmulti-organ failure or not), thrombotic thrombocytopenic purpura,thromboanginitis obliterans, or thrombotic disease associated withheparin induced thrombocytopenia, (e) the treatment or prevention ofthrombotic complications associated with extracorporeal circulation(e.g. renal dialysis, cardiopulmonary bypass or other oxygenationprocedure, plasmapheresis), (f) the treatment or prevention ofthrombotic complications associated with instrumentation (e.g. cardiacor other intravascular catheterization, intra-aortic balloon pump,coronary stent or cardiac valve), and (g) those involved with thefitting of prosthetic devices.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds disclosed hereinand practice the claimed methods. The following working examplestherefore, specifically point out preferred embodiments, and are not tobe construed as limiting in any way the remainder of the disclosure.

EXAMPLES Example 1 Production of 2-Ethxoycarbonyl-4-oxo-4H-benzopyran(Ethyl 4-Oxochromene-2-carboxylate)

In a mixture containing 180 g of toluene and 12.0 g of diethyl oxalatewas dissolved 30 g of 2-hydroxyacetophenone, to which 65.0 g of a 20%solution of sodium ethylate in ethanol was added dropwise. Aftercompletion of the reaction 13 g of 98% sulfuric acid was subsequentlyadded, and the mixture was stirred at 60° C. for about 30 minutes. Then140 g of water was added, and the mixture was subjected to separation ofthe organic layer. The resultant organic layer was concentrated, afterwhich 55.0 g of hexane was added and the mixture was filtered below 10°C. which yields about 34.0 g of ethyl 4-oxochromene-2-carboxylate.(Approximately 95% yield).

Example 2 Production of Ethyl 4-Oxochromane-2-carboxylate

A hydrogenator was charged by adding 6 g of ethyl4-oxochromene-2-carboxylate, 3.5 mL of acetic anhydride, 1 g of 10%palladium on carbon, 4.0 g of dried 3A molecular sieves (powered), and30 mL of glacial acetic acid. After purging several times with nitrogen,the hydrogenator was purged several times with hydrogen. Whilemaintaining stirring the reaction mixture was pressurized to about 30psi of hydrogen, heated to about 60° C. and maintained under thoseconditions for about 10-12 hours. HPLC monitoring of the reaction wasused to determine when the reaction was essentially complete, e.g., whenthe area ratio by HPLC between the chromen-4-one and the chroman-4-onewas not more than 3%. The mixture was then cooled to room temperatureand filtered through a celite bed. The catalyst and sieves were washedwith 10 mL aliquots of glacial acetic acid and the washes were combinedwith the filtrate. The combined mixture was concentrated under milddistillation conditions to an oil, which was dissolved with ethylacetate and extracted with saturated NaHCO₃. After the extraction, theaqueous layer was washed with ethyl acetate and the mixture neutralizedto a low pH with concentrated HCl. The mixtures was extracted severaltimes with ethyl acetate, the extracts were combined, concentrated to asolid, washed with acetonitrile and then filtered. Upon drying, about3.5-4.0 grams of ethyl 4-oxo-chromane-2-carboxylate were obtained as asolid.

Example 3 Production of Ethyl 6-Nitro-4-oxochromane-2-carboxylate

To a sulfuric acid solution of the ethyl 4-oxochromane-2-carboxylate ofExample 2 at −10° C. (about 3 ml) was added potassium nitrate insulfuric acid (about 1 ml), wherein the molar ratio of the potassiumnitrate starting material to the chromone compound starting material wasslightly in excess of 1:1. The reaction mixture was stirred at about 0°C. for 1 hour, the ice bath was removed and the reaction mixture wasstirred at room temperature for 46 hours. The nitrated chromone compoundforms a precipitate and the reaction was maintained at room temperatureuntil monitoring of the solution with HPLC shows the solution to beessentially free of the chromone starting material (less than 3%). Thereaction mixture was poured into ice and the precipitate was extractedwith ethyl acetate. The ethyl acetate layer was dried, filtered andevaporated to give a light yellow solid. The yield of ethyl6-nitro-4-oxochromane-2-carboxylate was approximately 80%.

Example 4 Production of Ethyl 6-Nitro-4-oxochromane-2-carboxylate

In a mixture containing 1 mole of 4-nitrophenol were added 2 moles ofdiethyl diester of maleic add and 1120 ml methane sulfonic acid. Themixture was then heated to 92° C. for 20 hours. The reaction was cooledto 0° C., poured onto 2 liters of ice and 2 liters of water andextracted with 3 times with 800 mls of diethyl ether. The organic layerswere combined, washed with 3×500 ml water, 4×500 ml 1N NaOH, 2×500 mlwater, and 500 ml brine, dried over magnesium sulfate and concentratedunder vacuum to yield about 50-60 g of crude yellow solid, which wasethyl 6-nitro-4-oxochromane-2-carboxylate. (Approximately 45%-65%yield).

Example 5 In Situ Ester Production of Ethyl6-Nitro-4-oxochromane-2-carboxylate

In a mixture containing 1 mole of 4-nitrophenol was added 2 moles ofmaleic acid and 500 mL of 1N sulfuric acid. The mixture was then heatedto 92° C. for 10-20 hours and monitored by HPLC for completion of thereaction with respect to the nitrophenol starting material. The reactionwas cooled to 50° C. and 2 moles of ethanol were added. The temperaturemaintained between 40° C.-55° C. for 1-3 hours or until HPLC indicatesthat the esterification was complete. The reaction was cooled to 0° C.,poured onto 2 liters of ice and 2 liters of water and extracted with 3times with 800 mls of diethyl ether (or ethanol). The organic layerswere combined, washed with 3×500 ml water, 4×500 ml 1N NaOH, 2×500 mlwater, and 500 ml brine, dried over magnesium sulfate and concentratedunder vacuum to yield about 75 g of crude yellow solid, which was ethyl6-nitro-4-oxochromane-2-carboxylate. (Approximately>60-75% yield).

Example 6 Production of Ethyl 2-(4-Oxo-chromen-2-yl) Acetate

In a mixture containing 1 mole of phenol was added 1.5 moles ofbeta-keto glutaric acid, 500 mL of 1N sulfuric acid and 200 mL ofethanol. The mixture was then heated to 80° C. for 10-20 hours andmonitored by HPLC for completion of the reaction with respect to thephenol starting material. The reaction was cooled to 0° C. and pouredonto 2 liters of ice and 2 liters of water and extracted with 3 timeswith 800 mls of diethyl ether (or ethanol). The organic layers werecombined, washed with 3×500 ml water, 4×500 ml 1N NaOH, 2×500 ml water,and 500 ml brine, dried over magnesium sulfate and concentrated undervacuum to yield about 60-70 g of a crude yellow solid, which was ethyl2-(4-oxo-chromen-2-yl) acetate. (Approximately>55-65% yield).

Example 7 Production of Ethyl 2-(6-Nitro-4-oxo-2-yl) Acetate

The crude mixture obtained in Example 6 was reduced using 10% palladiumon carbon substantially as set forth in Example 2, above, and thennitrated substantially as set forth in Example 3, above, to yield aracemate of ethyl 2-(6-nitro-4-oxo-chroman-2-yl) acetate.(Approximately>80% yield with respect to the amount of starting materialobtain from Example 6).

Example 8 Production of 6-Nitro-chromen-4-one

One gram molecular weight of 4-oxo-[4H]-benzopyran (Aldrich CatalogNumber 19,922-2), i.e., chromen-4-one, was nitrated and recovered usingthe procedures set forth in Example 3, above, to yield about 92 g of6-nitro-chromen-4-one, approximately 60% yield with respect to thestarting material.

Example 9 Production of Benzopyrilium Salt of 6-Nitro-chromen-4-one

The crude material of Example 8 was reacted with TBSOTf under standardsalt-forming conditions to yield the benzopyrilium salt in about 95%yield.

Example 10 Production of Ethyl 2-(4-[TBS-enol]-6-nitro-chroman-2-yl)Acetate

The benzopyrilium salt of Example 9 was reacted with a molar excess ofthe ketene enol resulting from TBSOTf reaction with the ethyl ester ofacetic acid, by adding the ketene enol dropwise to the benzopyriliumsalt dropwise to produce the product in a 90% yield based upon thebenzopyrilium salt starting material.

Example 11 Production of Ethyl 2-(6-nitro-4-oxo-chroman-2-yl) Acetate

The 4[TBS-enol] of Example 10 was converted to the ketone by acidify thereaction mixture with HCl and allowing the reaction mixture to come toroom temperature while stirring for 4-6 hours and the reaction mixturewas cooled over ice. The precipitate crystals were separated extractedwith ethyl acetate. The ethyl acetate layer was dried and filtered toyield a light yellow solid in about 85-90% yield with respect to thebenzopyrilium salt starting material of Example 9. The light yellowsolid was the racemate of ethyl 2-(6-nitro-4-oxo-chroman-2-yl) acetate.

Example 12 which follows provides a specific example of enzymaticresolution. However, this process may be used for compounds havingdifferent acid chain lengths and different ester groups, and may also beused for different types of lipase enzymes.

Example 12 Enymatic Resolution of Ethyl 2-(6-nitro-4-oxo-chroman-2-yl)Acetate Racemate

Approximately 330 g of the ethyl 2-(6-nitro-4-oxo-chroman-2-yl) acetateracemate of Example 11 was placed in isopropyl alcohol and water in thepresence of 1 g of the lipase enzyme ChiroCLEC-PC (Altus, Inc.) underthe conditions set forth in the ChiroCLEC-PC Information Bookletavailable from Altus for 24 hours. After separation of the free acidproduct from the ester substrate, approximately 160 gm of the2-2S6-nitro-4-oxochroman-2-yl-acetic acid was obtained in 98.5% purity.

Examples 13 and 14, which follow, provide a specific example ofconversion of a 6-nitro-4-oxo-chroman-2-yl add to a 6-amino-chroman-2-yladd ester salt. The procedures of these examples may be used forcompounds having esters other than ethyl and acid groups other thanacetic. In addition, adds and their corresponding esters as well assalts and their corresponding free bases may be interconverted usingmethods known to those skilled in the art.

Example 13 Production of 2-[2S) 6-Acetamido-chroman-2-yl] Acetic Acid

A hydrogenator was charged by adding 6 g of 2-[(2S)6-nitro-4-oxo-chroman-2-yl] acetic acid, 3.5 mL of acetic anhydride, 1 gof 10% palladium on carbon, 4.0 g of dried 3A molecular sieves(powered), and 30 mL of glacial acetic acid. After purging several timeswith nitrogen, the hydrogenator was purged several times with hydrogen.While maintaining stirring the reaction mixture was pressurized to about70 psi of hydrogen, heated to about 80° C. and maintained under thoseconditions for about 10-12 hours. The bomb was then cooled to about 50°C., evacuated of hydrogen and purged several times with nitrogen.Trifluroroacetic acid (3.5 mL) was added the bomb, the bomb wasresealed, was purged several times with hydrogen and was thenpressurized to 70 psi of hydrogen. The reaction mixture was stirred asit was heated to 80° C. and was maintained at 80° C. with stirring. HPLCmonitoring was used to determine when the reaction was essentiallycompleted (the area ratio by HPLC between the intermediate and productwas not more than 3%) and the mixture was cooled to room temperature.After filtering of the mixture through a celite bed, the catalyst andsieves were washed with 10 mL aliquots of glacial acetic acid and thewashes combined with the filtrate. The combined mixture was concentratedunder mild distillation conditions to an oil, which was dissolved withethyl acetate and extracted with saturated NaHCO₃. After the extraction,the aqueous layer was washed with ethyl acetate and the mixtureneutralized to a low pH with concentrated HCl. The mixtures wasextracted several times with ethyl acetate, the extracts were combined,concentrated to a solid, washed with acetonitrile and then filtered.Upon drying, about 3.5-4.0 grams of ethyl2-((2S)-6-acetamido-chroman-2-yl) acetate were obtained as a whitesolid.

Example 14 Production of Hydrochloride Salt of Ethyl 2-((2S)6-Amino-chroman-2-yl) Acetate

A mixture of 1.5 g of the ethyl 2-((2S)-6-acetamido-chroman-2-yl)acetate of Example 13, above, in 25 mL of concentrated sulfuric acid wasstirred vigorously at room temperature for about 6 hours, 50 mL ofethanol was added and the mixture was allowed to sit overnight. Theprecipitate was recovered by filtration and rinsed with 50 mL aliquotsof ether and dried. Absolute ethanol (25 mL) and HCl (10 mL) were mixedwith the precipitate for 2 hours followed by addition of 10 mL ofconcentrated HCl. The precipitate was recovered and recrystallized twicein an ether/isopropanol solvent mixture. Yielded was about 1.4 g ofethyl 2-((2S)-6-amino-chroman-2-yl) acetate hydrochloride (about 85-90%yield).

Example 15 Production of Ethyl 2-(6-Nitro-4-oxo-chroman-2-yl) AcetateRecemate

Approximately 330 g of the ethyl 2-((2R)-6-nitro-4-oxo-chroman-2-yl)acetate of Example 12 was separated in 99.4% purity from the (2S)isomer. To the (2R) enantiomer ester was added 500 mL of ethanol and acatalytic amount of potassium ethanolate (<1 equivalent). The mixturewas maintained at room temperature for 28 hours with stirring.Saponification was performed by addition of aqueous 1 N NaOH in ethanolfor 3 hours. After acidification with 1N aqueous hydrochloric acid the6-nitro-4-oxochroman-2-yl acetic acid racemate was recovered as aprecipitate in approximately 80% yield (about 250 g) with respect to the(2R) enantiomer ester starting material.

Example 16 Production of Ethyl 2-(6-Nitro-4-oxo-chroman-2-yl) AcetateRecemate

About 2.5 g of the crude precipitate of Example 15 was recovered andadded to 40 mL of concentrated sulfuric acid with vigorous stirring atroom temperature for about 6 hours, 75 mL of ethanol was added and themixture was allowed to sit overnight. The precipitate was recovered byfiltration and rinsed with 60 mL aliquots of ether and dried. Absoluteethanol (40 mL) and HCl (20 mL) were mixed with the precipitate for 2hours. The precipitate was recovered and recrystallized twice in anether/isopropanol solvent mixture. About 2.4 g of the ethyl2-(6-nitro-4-oxo-chroman-2-yl) acetate racemate was recovered (about85-90% yield), which can be recycled to Example 12 for enzymaticresolution of the enantiomers and a higher overall recovery of the (2S)enantiomer.

Examples 17-19 describe a specific procedure for lengthening a2-carboxyl chain on a chroman-4-one by one carbon. The procedure may beadapted to lengthen the chain by more than one carbon, including twocarbons and three carbons, by the use of appropriate reagents.

Example 17 Production of 2-(Hydroxymethy)-chroman-4-one

The carboxylic acid group of the compound of Example 2 (ethyl4-oxochromane-2-carboxylate) was treated with a metallic hydroxide basein ethanol, and then the aqueous layer was treated with an acid to formthe free acid, which was washed with water and then dried to yield 3 gof 4-oxo-chromane-2-carboxylic acid.

The 3 g of 4-oxo-chromane-2-carboxylic acid in THF (65 mL) was stirredwas cooled to 0° C. and borane-methyl sulfide complex (2.5 mL of Msolution, 25 mmoles) was added dropwise for about 15 minutes. Thesolution was warmed to room temperature and then heated at reflux for 4hours. The solution was cooled to room temperature and 10% aqueoushydrochloric acid (20 mL) was added over 15 minutes and the solution wasstirred at room temperature for 2 hours. The mixture was concentrated toapproximately 25 mL. The solution was poured into ethyl acetate (50 mL)and washed with water (2×30 mL), saturated sodium bicarbonate (2×30 mL)and saturated ammonium chloride (2×30 mL). The organic layer wasseparated dried over anhydrous MgSO4, and then concentrated in vacuo toyield about 2.6 g of 2-(hydroxymethyl)-chroman-4-one (86.7% yield)

Example 18 Production of 2-Cyanomethylchromen-4-one

To a solution of 2.0 g of 2-(hydroxymethyl)-chroman-4-one (from Example17) in 35 mL of CH₂Cl₂ and 1.5 mL of pyridine was added 2 gp-toluenesulfonylchloride. The mixture was stirred at 25° C. for 36hours, then diluted with 20 mL ether and washed with 10 mL. The organiclayer was dried over MgSO₄ and concentrated to give 3.4 grams of crudetosylate. To the crude tosylate in 20 mL of DMSO was added with stirring80 mg of powdered sodium cyanide and the mixture was heated to refluxfor 1.5 hours under an inert atmosphere. The cooled mixture was dilutedwith 50 mL of water and extracted with 6 100 mL portions of ether andthe ether extracts were dried over anhydrous MgSO4 and filtered. Thefiltrate was concentrated and the residue was recrystallized withether/isopropanol to yield 1.7 grams of2-cyanomethylchroman-4-one.(about 85% yield).

Example 19 Production of Ethyl 2-(4-Oxochroman-2-yl) Acetate

A mixture of 1.5 g of 2-cyanomethylchroman 4one (Example 20) in 25 mL ofconcentrated hydrochloric add was stirred vigorously at room temperaturefor about 6 hours, 50 mL of ethanol was added and the mixture wasallowed to sit overnight. The precipitate was recovered by filtrationand rinsed with 50 mL aliquots of ether and dried. Absolute ethanol (25mL) and HCl (10 mL) were mixed with the precipitate for 2 hours followedby addition of 10 mL of concentrated HCl. The precipitate was recoveredand recrystallized twice in an ether/isopropanol solvent mixture.Yielded was about 1.3 g of ethyl 2-(4-oxo-chroman-2-yl) acetate (about80% yield).

Example 20 Production of Ethyl 2-(6-Nitro-4-oxo-chroman-2-yl) Acetate,Racemate

The 1.3 g of the ethyl 2-(4-oxo-chroman-2-yl) acetate, racemate (fromExample 19) was nitrated using generally the procedures described inExample 3 to yield 1.04 g of ethyl 2-(6-nitro-4-oxo-chroman-2-yl)acetate (80% yield).

Example 21 Production of Ethyl 2-[(2S) 6-Nitro-4-oxo-chroman-2-yl]Acetate

The racemate of Example 20 was resolved into the respective enantiomersusing the procedures in Example 12 to separate out the (2S) enantiomer.Alternatively, the free acid was formed and an esterification wasperformed in the present of the lipase as describe in the ChiroCLEC-PCInformation Booklet from Altus for 24 hours and the (2R) enantiomer wasobtained instead of the (2S) enantiomer. Either enantiomer was obtainedin 98.5% purity.

Example 22 Production of Ethyl 2-(6-Nitro-4-oxo-chroman-2-yl) AcetateRacemate From an Enriched Ethyl 2-[(2R>2S) 6-Nitro-4-oxo-chroman-2-yl]Acetate Composition

Approximately 330 g of the ethyl ester of2-[(2R>2S)nitro-4-oxo-chroman-2-yl] acetic acid of Example 12 wasseparated in about 98 purity for the (2R) isomer from the (2S) isomer.To the (2R) enantiomer ester was added 500 mL of ethanol and a catalyticamount of potassium ethoxide (<1 equivalent). The mixture was maintainedat room temperature for 28 hours with stirring. Saponification wasperformed by addition of aqueous 1 N NaOH in ethanol for 3 hours. Afteracidification with 1N aqueous hydrochloric acid the racemic (2Sapproximately equal to 2R) 2-(6-nitro-oxo-chroman-2-yl) acetic acid wasrecovered as a precipitate in approximately 80% yield (about 250 g) withrespect to the (2R) enantiomer ester starting material. The ethyl2-(6-nitro-4-oxo-chroman-2-yl) acetate racemate was formed from the freeacid using the general esterification procedures as set forth in Example14, above, which can be recycled to Example 12 for enzymatic resolutionof the enantiomers and a higher overall recovery of the (2S) enantiomer.

The procedures in the above examples directed to resolution of the6-nitro substituted ethyl ester of the acetic acid (2S or 2R) chromanylenantiomer from the racemate, can readily be adapted to resolution ofthe acetamido derivatives of the same structures and to nitro positionisomers, as well as homologs of the compounds. For example, theenzymatic resolution procedures of Example 12, may be readily adapted tothe acetamido derivatives

The procedures above may be altered to use different starting materials,such as those having different substituents at the 6-position (amino,protected amino, hydrogen, etc.) Additionally, other minor modificationsmay be done, such as substitution of other known reagents, othercatalysts, etc. Furthermore, other alcohols may be used to make otheresters, or the esters may be hydrolyzed to provide the free acid.

In view of the above description it is believed that one of ordinaryskill can practice the invention. The examples given above arenon-limiting in that one of ordinary skill in view of the above willreadily envision other obvious permutations and variations withoutdeparting from the principal concepts embodied therein. Suchpermutations and variations are also within the scope of the disclosure.

What is claimed is:
 1. A process for making a compound, or a saltthereof, having a general formula:

wherein R is C₁-C₈ alkyl and n=0 to about 3, comprising: (a) reactingphenol and beta-keto glutaric acid in H₂SO₄ and ethanol with heat tocreate a reaction mixture, followed by pouring the reaction mixture ontoice water, extracting a chromenone product formed from (a) into anorganic solvent and evaporating the organic solvent as follows:

(b) hydrogenating the chromenone product from (a) above to produce acorresponding chromanone:

(c) nitrating the chromanone from (b) to create a racemic mixture asfollows:

(d) resolving the racemic mixture using a lipase enzyme, as follows:

(e) hydrogenating the product from (d) to convert the oxo group to amethylene group and convert the nitro group to an amino group andsubsequently protecting the amino group as an acetamido group asfollows:

(f) acidifying the product from (e) above to form an amine followed byaddition of concentrated HCl to the amine to produce an HCl salt of theamine as follows:


2. A process for making a compound, or a salt thereof, having a generalformula:

wherein R is C₁-C₈ alkyl and n=0 to about 3, comprising: (a) reacting2-hydroxyacetophenone and diethyloxalate in the presence of sodiumethoxide followed by addition of concentrated sulfuric acid to form achromen-4-one as follows:

(b) hydrogenating the chromen-4-one to form a chroman-4-one as follows:

(c) performing a chain extension on the product from (b) by firstforming a free acid from the ester, followed by reacting the free acidwith borane-methyl sulfide complex to form a 2-hydroxymethyl derivative,followed by replacing the hydroxy group with a tosyl group and reactingthe tosyl derivative with a cyanide salt to form a 2-cyano derivative,followed by hydrolyzing the cyano derivative in concentrated acid toform a 2-acid group and esterifying the 2-acid group as follows:

(d) nitrating the product from (c) above to form a 6-nitro group as partof a racemic mixture as follows:

(e) resolving the racemic mixture using a lipase enzyme, as follows:

(f) hydrogenating the product from (e) to convert the oxo group to amethylene group and convert the nitro group to an amino group andsubsequently protecting the amino group as an acetamido group asfollows:

(g) acidifying the product from (f) above to form an amine followed byaddition of concentrated HCl to the amine to produce an HCl salt of theamine as follows


3. A process for making a compound, or a salt thereof, having a generalformula:

wherein R is C₁-C₈ alkyl and n=0 to about 3, comprising: (a) reactingnitrophenol and diethyl ester of maleic acid with methane sulfonic acidunder heating as follows:

(b) performing a chain extension on the product from (a) by first makinga free acid from the ester, followed by the free acid reacting withborane-methyl sulfide complex to form a 2-hydroxymethyl derivative,followed by replacing the hydroxy group with a tosyl group and reactingthe tosyl derivative with a cyanide salt to form a 2-cyano derivative,followed by hydrolyzing the cyano derivative in concentrated acid toform a 2-acid group and esterifying the 2-acid group as follows:

(c) resolving the racemic mixture using a lipase enzyme, as follows:

(d) hydrogenating the product from (c) to convert the oxo group to amethylene group and convert the nitro group to an amino group andsubsequently protecting the amino group as an acetamido group asfollows:

(e) acidifying the product from (d) above to form an amine followed byaddition of concentrated HCl to the amine to produce an HCl salt of theamine as follows:


4. A process for making a compound, or a salt thereof, having a generalformula:

wherein R is C₁-C₈ alkyl and n=0 to about 3, comprising: (a) nitratingthe chromen-4-one at the 6-position as follows:

(b) reacting the product from (a) above with TBSOTf to form abenzopyrillium salt as follows:

(c) adding a ketene enol to the benzopyrillium salt from (b) above asfollows:

(d) acidifying the product from (c) above to remove the TBS group at the2-position on the 6-nitro-4-oxochromane ring to form a racemic mixtureas follows:

(e) resolving the racemic mixture using a lipase enzyme, as follows:

(f) hydrogenating the product from (e) to convert the oxo group to amethylene group and convert the nitro group to an amino group andsubsequently protecting the amino group as an acetamido group asfollows:

(g) acidifying the product from (f) above to recover an amine followedby addition of concentrated HCl to the amine to produce an HCl salt ofthe amine as follows:


5. A composition produced by a method of claim 1, comprising about 75%to about 100% of a single (2R) or (2S) enantiomer of 6-aminochroman-2-ylacetic acid or an ester thereof.
 6. A process according to claim 1wherein the enzyme is a lipase from Pseudomonas cepacia.
 7. A processaccording to claim 6 wherein the lipase is ps 30 lipase.
 8. A processaccording to claim 1, wherein the lipase is a lipase stabilized bycross-linking with alpha keto glutarate.
 9. A process according to claim8, wherein the enzyme is stabilized ps 30 enzyme ChiroCLEC-PC.
 10. Acomposition produced by a method of claim 2, comprising about 75% toabout 100% of a single (2R) or (2S) enantiomer of 6-aminochroman-2-ylacetic acid or an ester thereof.
 11. A process according to claim 2wherein the enzyme is a lipase from Pseudomonas cepacia.
 12. A processaccording to claim 11, wherein the lipase is PS 30 lipase.
 13. A processaccording to claim 2, wherein the lipase is a lipase stabilized bycross-linking with alpha keto glutarate.
 14. A process according toclaim 13, wherein the enzyme is stabilized PS 30 enzyme ChiroCLEC-PC.15. A composition produced by a method of claim 3, comprising about 75%to about 100% of a single (2R) or (2S) enantiomer of 6-aminochroman-2-ylacetic acid or an ester thereof.
 16. A process according to claim 3wherein the enzyme is a lipase from Pseudomonas cepacia.
 17. A processaccording to claim 16, wherein the lipase is PS 30 lipase.
 18. A processaccording to claim 3, wherein the lipase is a lipase stabilized bycross-linking with alpha keto glutarate.
 19. A process according toclaim 18, wherein the enzyme is stabilized PS 30 enzyme ChiroCLEC-PC.20. A composition produced by a method of claim 4, comprising about 75%to about 100% of a single (2R) or (2S) enantiomer of 6-aminochroman-2-ylacetic acid or an ester thereof.
 21. A process according to claim 4wherein the enzyme is a lipase from Pseudomonas cepacia.
 22. A processaccording to claim 21, wherein the lipase is PS 30 lipase.
 23. A processaccording to claim 4, wherein the lipase is a lipase stabilized bycross-linking with alpha keto glutarate.
 24. A process according toclaim 23, wherein the enzyme is stabilized PS 30 enzyme ChiroCLEC-PC.